Mutations in BRCA1 and BRCA2, the first two breast cancer susceptibility genes identified, carry as much as an 85% lifetime risk of developing breast, ovarian or other cancers. Genetic testing for mutations in these two genes has recently become commercially available. There have been varying amounts of psychological distress noted among women with a family history of breast cancer. Distress has been observed to impact psychological functioning, activities of daily living, and the practice of breast cancer surveillance behaviors. Within the genetic screening process, psychological distress has been shown to impact the decision to undergo genetic screening, the comprehension and retention of risk assessment information, as well as affecting the subject following the receipt of the genetic test results. Little work has been done to examine predictors of distress within at risk subjects. This study examines psychological distress among 52 community women presenting for BRCA1 and BRCA2 genetic mutation testing. Predictors of distress included family cancer history, education, age, Ashkenazi ethnicity, and Internality and Powerful Others Health Locus of Control. Vulnerable sub-groups of patients include younger women, women with higher levels of education and women of Ashkenazi ethnicity.

We attempted to understand the molecular regulators that impact inflammation using a rat model of human sensation-seeking/risk-taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress. We found that HRs have a pro-inflammatory phenotype as indicated by increased protein expression of the inflammatory cytokine TNF-(Sa(B. Furthermore, we found that HRs have a lower gene expression of the glucocorticoid receptor and histone deacetylase 2 which are known to play an immunosuppressive role. Autophagy (macroautophagy) is a homeostatic process needed for cell maintenance, growth and proliferation and known to assist in tumor survival. FYVE and coiled-coil domain containing 1 (FYCO1) is a novel protein implicated to assist in the plus-end directed trafficking and fusion of autophagosomes. In these studies, we show that FYCO1 gene expression among human breast cell lines of varying degrees of malignancy. / Lillian C. Onwuka-Ekpete. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Mak, Ko Fung. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 113-138). / Abstracts in English and Chinese. / Chapter CHAPTER I: --- INTRODUCTION --- p.1 / Chapter I. --- AIM OF STUDY --- p.1 / Chapter II. --- LITERATURE REVIEW --- p.2 / Chapter 1. --- Epidemiology --- p.2 / Chapter 2. --- Etiology --- p.4 / Chapter A. --- Heredity --- p.4 / Chapter i. --- Family History --- p.4 / Chapter ii. --- Inherited Predisposition --- p.4 / Chapter B. --- Hormonal --- p.7 / Chapter C. --- Environmental --- p.9 / Chapter i. --- Diet --- p.9 / Chapter ii. --- Radiation --- p.10 / Chapter iii. --- Physical Activity --- p.11 / Chapter 3. --- Histopathology --- p.12 / Chapter 4. --- Clonality Nature of Cancer --- p.13 / Chapter 5. --- "Knudson ""two-hit"" Hypothesis on Cancer Development" --- p.14 / Chapter 6. --- Molecular Genetic Studies of Breast Cancer --- p.15 / Chapter A. --- Loss of Heterozygosity --- p.16 / Chapter B. --- Comparative Genomic Hybridization --- p.19 / Chapter C. --- Epigenetic Changes --- p.20 / Chapter 7. --- Genetic Changes in Breast Cancer --- p.21 / Chapter A. --- Chromosome 1 --- p.21 / 14-3-3σ Gene --- p.21 / Chapter B. --- Chromosome 3 --- p.22 / Chapter i. --- Retionoic Acid Receptor p2 Gene --- p.22 / Chapter ii. --- Fragile Histidine Traid Gene --- p.24 / Chapter iii. --- Ras Associated Domain Family 1A Gene --- p.25 / Chapter iv. --- Thyroid Hormone Receptor β1 Gene --- p.26 / Chapter C. --- Chromosome 5 --- p.27 / Adenomatous Polyposis Coli Gene --- p.27 / Chapter D. --- Chromosome 6 --- p.28 / Estrogen Receptor Gene --- p.28 / Chapter E. --- Chromosome 9 --- p.29 / p16 Gene --- p.29 / Chapter F. --- Chromosome 13 --- p.30 / Chapter i. --- BRCA2 Gene --- p.31 / Chapter ii. --- Retinoblastoma Gene --- p.32 / Chapter G. --- Chromosome 16 --- p.33 / E-cadherin Gene --- p.33 / Chapter H. --- Chromosome 17 --- p.34 / Chapter i. --- TP53 Gene --- p.34 / Chapter ii. --- BRCA1 Gene --- p.36 / Chapter CHAPTER II: --- MATERIALS AND METHODS --- p.38 / Chapter I. --- PATIENTS AND SPECIMENS --- p.38 / Chapter II. --- FROZEN SECTIONS AND MICRODISSECTION --- p.41 / Chapter III. --- DNA EXTRACTION --- p.43 / Chapter IV. --- ALLELOTYPING --- p.44 / Chapter 1. --- Polymerase Chain Reaction --- p.44 / Chapter 2. --- Electrophoresis --- p.45 / Chapter 3. --- Data Analysis --- p.46 / Chapter CHAPTER III: --- RESULTS --- p.49 / Chapter I. --- ALLELOTYPING ANALYSIS --- p.49 / Chapter II. --- FREQUENCY OF LOH --- p.68 / Chapter III. --- FRACTIONAL ALLELIC LOSS --- p.70 / Chapter IV. --- MINIMAL DELETION REGIONS --- p.72 / Chapter 1. --- Chromosome 6q --- p.83 / Chapter 2. --- Chromosome 8p --- p.83 / Chapter 3. --- Chromosome 8q --- p.84 / Chapter 4. --- Chromosome 10q --- p.84 / Chapter 5. --- Chromosome 13q --- p.85 / Chapter 6. --- Chromosome 14q --- p.85 / Chapter 7. --- Chromosome 16q --- p.86 / Chapter V. --- MICROSATELLITE INSTABILITY --- p.86 / Chapter CHATPER IV: --- DISCUSSION --- p.88 / Chapter I. --- COMPARISONS OF CHROMOSOMAL ALTERATIONS --- p.88 / Chapter II. --- MICROSATELLITE INSTABILITY --- p.92 / Chapter III. --- CHROMOSOMAL GAINS AND LOSSES --- p.93 / Chapter IV. --- CHROMOSOME 17 --- p.95 / Chapter V. --- MINIMAL DELETION REGIONS --- p.96 / Chapter 1. --- Chromosome 6q --- p.97 / Chapter 2. --- Chromosome 8p --- p.99 / Chapter A. --- 8p23.3.-22 --- p.99 / Chapter B. --- 8p21.3-12 --- p.101 / Chapter C. --- 8p22-21 --- p.103 / Chapter 3. --- Chromosome 8q --- p.104 / Chapter 4. --- Chromosome 10q --- p.106 / Haploinsufficiency and PTEN --- p.107 / Chapter 5. --- Chromosome 13q --- p.108 / Chapter 6. --- Chromosome 14q --- p.109 / Chapter 7. --- Chromosome 16q --- p.110 / Chapter CHAPTER V: --- CONCLUSION --- p.112 / REFERENCES --- p.113

Background. B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) acts as an oncogene in various cancer such as non-small cell lung cancer, colon cancer, gastric cancer, bladder cancer and nasopharyngeal cancer (NPC). / Methods. Immunohistochemistry was performed to evaluate Bmi-1 expression in 252 breast cancer samples. The correlations were analyzed between Bmi-1 expression and clinicopathologic parameters, including age, tumor size, lymph nodal involvement, distant metastasis, clinical stages, hormone receptor (ER, PR) and Human Epidermal Growth Factor Receptor 2 (HER-2). The overall survivals were compared by Kaplan-Meier analysis based on Bmi-1 expression. / Results. Bmi-1 expression was significantly increased in primary cancer tissues than in matched adjacent non-cancerous tissues ( P<0.001). Only 35.9% (14 of 39) of adjacent non-cancerous tissues displayed high expression compared with 72.2% (182 of 252) in primary cancer tissues. Among adjacent non-cancerous tissues, no Bmi-1 staining signal was detected in 30.8% (12 in 39) samples. Only 28.2% (11 in 39) samples showed nucleus staining and the remaining 41.0% (16 in 39) samples exhibited cytoplasm staining. Of those cancer tissues, however, 75.4% (190 in 252) was stained in the nucleus and 24.6% (62 in 252) located in the cytoplasm. The elevated Bmi-1 expression was correlated with advanced clinicopathologic classifications (T, N, M) and clinical stages (P<0.001, respectively). A high level of Bmi-1 expression displayed unfavorable overall survival ( P<0.001). The overall survival rate, assessed by the Kaplan-Meier method, was 85.1% (57 in 67) in low Bmi-1 expression group, whereas it was only 59.9% (103 in 172) in high Bmi-1 expression group. In addition, Bmi-1 serves as a high risk for breast cancer and the relative risk increased almost four fold in patients with high Bmi-1 expression compared with that with low Bmi-1 expression by univariate Cox regression analyses. After the adjustment of the confounding factors, Bmi-1 was still found to predict the poor survival (P=0.042), which indicated Bmi-1 was an independent prognostic factor. The overexpression of Bmi-1 increased the mobility and invasiveness in 76N-TERT and MCF-10A, concurrent EMT-like molecular changes, the stabilization of Snail protein and the activation of Akt/GSK3beta pathway. Consistent with these observations, the repression of Bmi-1 in MDA-MB-435S remarkably attenuated the cellular mobility, invasiveness and transformation, as well as tumorigenesis and spontaneous lung metastases in nude mice. In addition, the repression of Bmi-1 reversed the EMT markers and inhibited the Akt/GSK3beta/Snail pathway. However, ectopic Bmi-1 alone was not able to lead to the phenotype of HMECs. Additionally, discordant mRNA expression levels of Bmi-1 and E-cadherin were detected between primary cancer tissues and matched adjacent non-cancerous tissues. The mRNA level of Bmi-1 was strongly up-regulated in breast cancer tissues compared with paired non-cancerous tissues ( P=0.001), whereas the mRNA level of E-cadherin was markedly down-regulated (P=0.042). Furthermore, there was a converse correlation between Bmi-1 and E-cadherin expression at the transcriptional level ( P=0.041). (Abstract shortened by UMI.) / Guo, Baohong. / Adviser: Kung, Hsiang Fu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 161-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Breast cancer is the most common malignancy in women and represents one of the major causes of death worldwide. The PI3K-Akt-FOXO3a signalling pathway has been shown to play a crucial role in tumorigenesis and the development of drug resistance in many cancer types. However, previous studies on FOXO3a using breast cancer tissues were controversial. So this study aims at better understanding of the role of FOXO3a in the development of drug resistance, especially endocrine resistance and anthracycline resistance in breast cancer. Examination of FOXO3a and phosphorylated-Akt (P-Akt) expressions in breast cancer tissue microarrays revealed nuclear FOXO3a was significantly associated with poor prognosis (p=0.014) and lymph node positivity (p=0.052) in invasive ductal carcinoma. Using the tamoxifen and anthracycline-sensitive and -resistant breast cancer cell lines as models, we found that the nuclear accumulation of FOXO3a was associated with enhanced anthracycline-resistance but not tamoxifen-resistance. This was consistent with the finding that sustained nuclear FOXO3a was associated with poor prognosis, as cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis. We demonstrated a possible feedback mechanism in which induction of FOXO3a activity in the anthracycline-sensitive MCF-7 cells induced Akt phosphorylation and promoted cell proliferation arrest. Using MDA-MB-231-FOXO3a(A3):ER cells in which FOXO3a activity could be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction could up-regulate PI3K-Akt activity but had little effect on cell proliferation, which indicates impaired Akt-FOXO3a axis in chemoresistant cell models. To further uncover the precise mechanism of Akt-FOXO3a deregulation in the development of chemoresistance, we have explored the post-translational regulation of FOXO3a by miRNAs. Through a series of Gain-and-Loss functional experiments and luciferase reporter assays in vitro, three miRNAs, including miR-222, miR-221 and miR-29a, were found to suppress FOXO3a protein expression through binding directly to FOXO3a 3’UTR. Moreover, the aberrant expressions of the miR-222/221 cluster and miR-29a in drug resistant cell lines could confer a proliferation advantage to cancer cells through suppressing FOXO3a expression. We further demonstrated that FOXO3a as a transcription factor could transactivate the oncogenic miR-222 and miR-221 expressions under certain chemotherapy stimulation. This suggests the existence of a feedback regulatory loop composed of the miR-222/221 cluster and FOXO3a which may not only play a self-protective role under drug treatment in chemosensitive cells, but also partially explain the tolerated nuclear FOXO3a in the breast cancer with poor prognosis. Taken together, our study suggested that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signalling axis, as in these breast cancers the nuclear-located FOXO3a was unable to induce cell death or cell cycle arrest. We also demonstrated post-translational regulation of FOXO3a by miR-222/221 and miR-29a, while aberrant expressions of miR-222/221 and miR-29a may promote cell resistance to therapy through directly suppressing FOXO3a. FOXO3a could further contribute to the deregulation of the miR-222/221 cluster as a transcription factor in breast cancer. Studying this Akt-FOXO3a-miRNAs signalling circuit will provide us better understanding in predicting and monitoring treatment response in breast cancer and other malignancies. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Tamoxifen is the most widely used endocrine therapy for both early and advanced estrogen receptor (ER) positive breast cancer patients. About half of the patients that initially respond to the antiestrogen become estrogen-independent and ultimately develop resistance to the treatment. The precise molecular mechanisms of tamoxifen resistance remain poorly understood. Dysregulation of microRNAs (miRNAs) has been frequently reported in breast cancer and linked to cancer development, progression and therapeutic response. To gain a more comprehensive picture of the miRNA regulatory network for modulating tamoxifen responsiveness, we examined global expression profiles of more than 600 miRNAs in a matched pair of tamoxifen-sensitive ZR75 and tamoxifen-resistant AK47 breast cancer cell lines using TaqMan Low Density Array (Applied Biosystems). Under 4-hydroxytamoxifen treatment, 102 miRNAs displayed differential responses between the sensitive cells and the resistant cells. At basal levels, upregulation of 32 miRNAs and downregulation of 75 miRNAs were observed in the resistant cells as compared to the sensitive cells. Among the 9 miRNAs of significant differential expression selected for validation, expression profiles of the 7 miRNAs could be reproduced. Of these, 4-hydroxytamoxifen treatment greatly increased miR-449a/b expression in sensitive ZR75 cells, whereas miR-449a/b expression was significantly reduced in resistant AK47 cells at basal levels, which was further confirmed in a panel of tamoxifen-resistant breast cancer cell lines. Such downregulation of miR-449a/b in the resistant cells was partially attributed to DNA methylation-mediated repression of miR-449a/b. Notably, miR-449a/b expression exhibited a significant positive correlation with ER-α status (miR-449a: P=0.006, miR-449b: P=0.013) and progesterone receptor (PR) status (miR-449a: P=0.010, miR-449b: P=0.021), and a prominent inverse association with tumor grade in 61 breast cancer tissues (miR-449a: P=0.001; miR-449b: P=0.009). Also, breast cancer patients with high miR-449a/b expression tended to have increased disease-free survival (miR-449a: P=0.019; miR-449b: P=0.117). To further support the tumor suppressor function of miR-449, stable miR-449b overexpression in the resistant cells reduced cell proliferation. More intriguingly, restoring miR-449b expression increased sensitivity to 4-hydroxytamoxifen-induced apoptosis via suppression of AKT activity without restoring ER-α expression. In contrast, miR-449a/b knockdown reduced ER-α and PR expression, but enhanced phosphorylation of AKT, extracellular signal-regulated kinase- 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and also ER-α at serine 167 and serine 118 residues. Furthermore, we demonstrated c-Myc is a target gene of miR-449 as confirmed by bioinformatics and experimental analyses. Computational algorithms predicted a highly conserved miR-449a/b binding site within C-MYC 3’untranslated region (3’UTR). Compared to the parental sensitive cells, c-Myc was overexpressed in the resistant cells. Forced expression of miR-449b suppressed c-Myc protein level. To further support the notion that c-Myc is a direct target of miR-449, interactions between miR-449b and C-MYC 3’UTR were confirmed by co-expression of miR-449b and c-Myc expression constructs and luciferase reporter assay. Taken together, our data strongly suggest the critical role of miR-449 in modulating altering response to tamoxifen via targeting c-Myc. Suppression of miR-449 repressed genomic ER action and concomitantly activated non-genomic ER pathways. These findings may provide insights to improve breast cancer management and open a wide avenue for therapeutic interventions for overcoming tamoxifen resistance. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Breast cancer is one of the most prevalent cancers affecting women worldwide. In the breast, estrogen receptor alpha (ERα), upon binding with ligands, activates gene transcription and promotes cell growth and proliferation. Tamoxifen, a selective antagonist of ERα in breast, has been proved to be effective therapeutically. In spite of this, resistance remains a prominent issue and underlying mechanisms are not yet fully understood. Aberrant regulation of ER expression at genetic and transcriptional levels has been implicated as the mechanisms accounting for tamoxifen resistance. However, regulation of ERα expression at translational level including protein synthesis and degradation has not yet been characterized and its relevance to tamoxifen resistance has not been described. At level of protein synthesis, eukaryotic translation initiation factor 4E (eIF4E) selectively enhances the translation of 4E-sensitive mRNAs which contain long and complex 5’-untraslated regions (5’-UTR). eIF4E is often over-expressed in cancers. In silico analysis revealed that ERα contained a highly structured 5’-UTR similar to reported eIF4E-sensitive mRNAs, suggesting that ERα mRNA might be eIF4Esensitive. We showed by polysome fractionation and subsequent Q-PCR quantification that the ERα mRNAs were more actively translated in the cell line expressing higher levels of eIF4E. Consistently, transient transfection of eIF4E into an ERα-positive cell line resulted in enhanced protein expression of ERα. Moreover, subcelluar fractionation showed that eIF4E was bound with ERα mRNAs in the nucleus thus participating in transportation of mRNAs from the nucleus into the cytoplasm. Therefore, eIF4E could positively modulate protein synthesis of ERα by enhancing mRNA export in the nucleus as well as translation in the cytoplasm. Their positive correlation was validated in vivo using 106 Chinese breast cancer samples (Chi-square test, p=0.004). It was also found that elevated expression of eIF4E could mediate resistance to tamoxifen treatment and enhance cell survival. This could be due to enhanced expression of ERα or activation of PI3K/Akt pathway upon eIF4E over-expression. At the level of degradation, ERα is conjugated to poly-ubiquitin chains catalyzed by multiple enzymes and degraded by 26S polysomes. Carboxyl-terminus of Hsc70- interacting protein (CHIP) is an E3 enzyme specific for ERα degradation through interaction with ERα’s ligand-binding domain (LBD). Various splicing variants of ERα have been reported and implicated in tamoxifen resistance by interfering with functions of ERα wild type. Variants ERαΔ4, ERαΔ5, ERαΔ6/7 and ERαΔ7 with different degrees of truncation in their LBDs and differential expression were detected or reported in human breast cancers. Their interactions with CHIP may be different, resulting in variations in degradation. We found that the degradation of ERαΔ6/7 through ubiquitin-proteasome pathway was impaired whilst the degradation of other variants were less affected. This finding suggests that the binding site of CHIP to ERαmight be located within the peptide sequences encoded by exon6. Furthermore, as ERαΔ6/7 plays a dominant negative role in regulating functions of ERα wild type, aborted degradation of this variant may result in accumulation of this variant in the cell, inhibiting and inactivating ERα, making the cells refractile to tamoxifen treatment. / published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

Heterozygous mutations in BRCA1 or BRCA2 predispose carriers to an increased risk for breast or ovarian cancer. Both BRCA1 and BRCA2 (BRCA1/2) play an integral role in promoting genomic stability through their respective actions during homologous recombination (HR) mediated repair and stalled replication fork protection from nucleolytic degradation. SMARCAD1 (SD1) is a SWI/SNF chromatin remodeler that has been implicated in promoting long-range end resection and contributes to HR. Using human cell lines, we show that SMARCAD1 promotes nucleolytic degradation in BRCA1/2-deficient cells dependent on its chromatin remodeling activity. Moreover, SMARCAD1 prevents DNA break formation and promotes fork restart at stalled replication forks. These studies identify a new role for SMARCAD1 at the replication fork. In addition to the work presented here, I discuss a method for introducing stop codons (nonsense mutations) into genes using CRISPR-mediated base editing, called iSTOP, and provide an online resource for accessing the sequence of iSTOP sgRNASs (sgSTOPs) for five base editor variants (VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, and SaKKH-BE3) in humans and over 3 million targetable gene coordinates for eight eukaryotic species. Ultimately, with improvements to CRISPR base editors this method can help model and study nonsense mutations in human disease.